Method for determining calciotropic activity of a preparation of Padina pavonic algae

ABSTRACT

A method of determining calciotropic activity of a preparation of algae of the genus  Padina pavonica.  The algae preparation may be in the form of a powder or an extract. The method consists of culturing human or animal osteoblasts in a culture medium containing calcium ions in a well plate. The preparation is added thereto and non-fixed calcium ions are eliminated in the culture medium. Further, the culture medium is acidified to destroy an extracellular matrix formed by the osteoblasts in the culture medium. Thereafter, filtering of the culture medium is carried out and then a concentration of fixed calcium ions in the osteoblasts is determined and compared with a calibration scale of a known activator of the fixation of calcium or in the presence of an inhibitor of the fixation of calcium or both.

PRIOR APPLICATION

This application is a division of U.S. patent application Ser. No.09/947,898 filed Sep. 6, 2001, now U.S. Pat. No. 6,811,999.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the field of chemistry and moreparticularly to that of plant chemistry.

More especially, it has as its aim a new process for obtaining atitrated preparation based on algae with active principles ensuring thefixing of calcium by the osseous cells.

Its specific aim is a process for obtaining powders or extracts from thealga Padina pavonica, titrated with active principles by means of acoefficient of equivalence with estradiol, which consists of dosing theextract or the powder of Padina pavonica using a method in whichosteoblasts are cultivated in a medium rich in calcium ions, an analysisis made of the calcium fixed by the cells of the extracellular matrix inthe presence or absence of agents antagonistic to the fixing of calcium,and the results are expressed either in arbitrary or international unitsin relation to a reference standard or by reference to the knownactivity of fixed doses of estradiol or of any other agent favouring thefixing of calcium.

DETAILED DESCRIPTION OF THE INVENTION

More precisely, the process according to the invention consists ofpreparing a suspension of a human or animal osseous cell line, byadjusting the cellular density determined by turbidimetry or bycalculating from a reference value, leaving to incubate for one to threedays at 37° C. in the presence of carbon dioxide and of a completeculture medium (CCM), adding to some wells an agent inhibiting thefixing of calcium or a calciotropic hormone, and some extract to betitrated, eliminating the supernatants from the culture, rinsing out thewells with a rinsing medium without calcium or magnesium (P.B.S. forexample) in such a way as to eliminate the non-fixed calcium from theculture medium, eliminating the supernatant fractions, and adjusting thevolume of the dispersion with a fixed volume of mineral acid such ashydrochloric acid, sulphuric acid, phosphoric acid or perchloric acid orwith any other product capable of destroying the extracellular matrixbuilt up by the cells being cultured. In conclusion, the mixture isadjusted and the calcium is dosed, for example according to the standardAFNOR no. NT-690.005.

The dosage of the calcium constitutes an indicator of activity which iseasy to determine. The fixing of the calcium by osteoblasts or cellsfrom a cell line is dependent on the calciotropic agents activating thefixing of calcium (estradiol, vitamins D3, calcitonin) or agentsdeleterious to the fixing of calcium such as inhibitors of the calciccanals (verapamil, cinchonine or diltiazem), or pro-inflammatorycytokines: prostaglandins, interleukins, PAF etc.

The extracts or powders of the algae Padina pavonica have the effect ofstimulating biological activity (synthesis of proteins andglycoaminoglycanes) but also act to restore physiological functions suchas the fixing of calcium by osteogenic cells. For this, the fixedcalcium is dosed with a fixed cell line, such as for example the cellline UMR 106 or the line G 292, in the presence or not of deleteriousagents such as inhibitors of the calcic canals and pro-inflammatoryagents such as IL 1. Normal osteoblasts give good results, but theiravailability presents a problem. The preparation to be analysed istested in parallel on the same cell line, in the presence or not ofthese same deleterious agents. The quantity of fixed calcium in relationto that obtained with the calciotropic agent and with those obtained inthe presence of agents inhibiting the fixing of calcium are compared.

In fact, it has been found that the active molecules of the alga Padinapavonica (MAPP) kept their property of improving the fixing of calciumby the osseous cells, even in the presence of an inhibitor of the calciccanals or an inflammatory agent such as interleukin IL-1. This indicatesthat the extract of Padina pavonica possesses one or more activeprinciples whose mode of action is different from that of knownsubstances which are not capable of improving the fixing of calcium inthe presence of a calcic inhibitor.

The difficulty of the situation comes from the fact that the activity ofthe alga varies greatly, depending on the season, the growth and thestage of development of the plant, the depth, the luminosity and thefact that the technician or final consumer wants a product whoseactivity is constantly. It is necessary therefore to produce an extractin conditions such that it is possible to be free from the variations inactive principles in the plant, and the problems arising such as—whatshould be the activity of the extract and how obtain a product with atitre that is always constant?

The present invention aims to solve the technical problem outlinedabove.

The harvesting of plants, their treatment and the manufacture ofstabilised extract require the dosage of the activity in the substancestreated. The gathering-in of the plant should be carried out when theactive principle content is at its optimum level, so it is necessary todetermine the moment. The preservation of plants before theirprocessing, the extraction of the raw material, the manufacture of theextract make it necessary to have at one's disposal an analytical methodmaking it possible to quantify the activity of the material.

The production of an extract usually calls for chemical dosage of theactive molecule. When the activity is brought about by a family ofmolecules, it becomes difficult or impossible to refer to a chemicaldosage. The difficulty of the problem comes from the fact that themolecules can exist in the form of isomers or tautomers, some active andsome inactive. If the isomerism is supported by functional groups, it ispossible to dose the functional groups and these support the totalityand the exclusivity of the activity of the active principles. We canillustrate this by citing the dosage of anthraquinones with senna,alkaloids, etc. In the case of cardiotonic heterosides of digitalis,anomalies of correlation between the chemical analysis and biologicalactivity have been observed many times, as a result of the multiplicityof the active principles with unequal levels of activity.

If the isomerism is supported essentially by structural elements, theanalysis will be more delicate and will require the characteristicstructural elements to be disclosed by spectrophotometric measurement,or call for characteristic reactions such as coloration of the functionsthat may be representative of the molecular family being researched. Forexample, in the case of cis-trans isomerism, the double link should befunctionalized by fixing a reactional function. On this function, itwill be possible to fix a chromophore group, by a bonding reaction(isothiocyanate of fluorescein, for example). It is also possible to usemass spectometry but it should be remembered that this technique isdestructive, and it will not then be possible to re-verify the activityof the fraction isolated.

In the case of numerous substances, it is usual to dose the activity bybiological means, for example in the case of penicillin, numerousvitamins such as vitamin A. vitamin E, antibodies, antigens, certainhormones such as insulin, FSH, GSH, cytokins such as interferon, TNFetc. This technique is applied essentially to dosages which would beimpossible by chemical means. The case of vitamins A and E provide agood illustration, since they exist in the form of numerous isomers.

The problem becomes even more complex when dealing with substanceswithout a chromophore group that is easily detectable and whosestructure is unknown. In this case, the dosage of the biologicalactivity seems to be the sole method or at least the method of choice.

Amongst the methods of dosage of activity, one finds methods which applyto substances modifying a biological activity of procaryotic oreucaryotic cells. The dosage of biological activity poses the problem ofthe stability of normal cells or cell lines. Furthermore, the signalanalysed may vary according to the operational criteria impossible tospecify in many cases. At the time of the usage of these methodologies,the authors are confronted with the variability of response of thecells. The response of the strain should therefore be calibrated inrelation to a reference system. This system does not give a goodresponse in the case in question, as it so happens that Padina is theonly known plant capable of inducing a response on the fixing of calciumin the presence of a calcic inhibitor (Inh-Ca) and in the presence of aninflammatory agent such as IL-1. All the other substances testedhitherto cannot restore the fixing of calcium in the presence of acalcic inhibitor (Inh-Ca).

After numerous technical difficulties, the Applicants have managed toproduce a dosage method that is efficient and reproducible.

The material to be tested is dosed on a cell culture. An analysis iscarried out of the calcium fixed by the osseous cells. To do this, thecalcium fixed by an osteoblastic cell line is dosed. In a non-limitingway, UMR 106 and G292, in the presence or not of deleterious agents suchas the Inh-Ca and the IL-1 may be cited. The preparation to be analysedwill also itself be tested, on the same cell line in the presence or notof agents such as Inh-Ca and IL-1.

In these same conditions, calcium fixed by the same cell line in thepresence of estradiol is dosed and a calibration scale is produced,expressed in weight of estradiol This calibration scale ranges from 10⁻³to 10⁻¹⁰ mol of estradiol or more frequently between 10⁻⁷ and 10⁻⁹ molof estradiol. In this way, it is possible to establish a correlationbetween the activity of the extract of Padina and an active quantity ofestradiol. We should stress that this comparison is made between theactivity of estradiol directly on the cell culture with the activeextract of Padina in the presence of deleterious agents such as anInh-Ca or IL-1. So it is not a true weight comparison and it is proposedthat the result should be expressed in Units of Activity (UA). A unit ofactivity is defined as the quantity of extract capable of increasing by50% the quantity of calcium fixed by 400,000 cells cultivated for 48hours in the presence of 1 ng of interleukin 1 per 1 ml well. The IL-1can be replaced by another agent inhibiting the fixing of calcium at anequi-active dose.

The extracts of Padina have been described, in particular in theEuropean patent application EP 0.655.250. The algae is then depletedafter drying, by a hydrocarbonated solvent such as cyclohexane oracetone, the vegetable matter is separated, and the extracted product isfiltered then evaporated until dry. A quantity of dry extract weighedprecisely is then dissolved in ethanol. Determining its activity is theobject of the present invention. In concrete terms, the processaccording to the invention consists of preparing a suspension of cultureof the human cell line G292 (ECAC no. 901-110522), adjusting the celldensity to 400,000 cells per ml of complete culture medium (CCM),supplemented by 10% of fetal calf serum. The cell suspension isdistributed at the rate of 1 ml per well on plates of 24 wells. It isleft to incubate overnight in an atmosphere of CO₂ at 37°. Thesupernatants are separated then dilution is carried out by a newaddition of CCM. An aliquot fraction of IL-1 is prepared and 5 μl ofinterleukin IL1 solution is added, namely 1 ng per well in the wells forthe cells which receive only one inhibiting treatment per IL-1.

For each trial of solution to be titrated, 5 μl of solution of Padinaextract is added. It is then left to incubate for the length of timenecessary to carry out the trial, normally between 24 and 72 hours, andpreferably 48 hours. Then the supernatants are eliminated and thecellular waste lumps rinsed three times with buffer PBS without calciumand without magnesium, previously heated to a temperature of 37° C., soas to eliminate all the calcium in solution brought about by the culturemedium. After the usual purification processes, the solution is mixedwith an acid and the calcium is dosed according to the method defined bythe standard AFNOR no. NF-T-90.005. The dosages of calcium are onlyindicators of activity and can be expressed in arbitrary units. Theyonly have absolute value if the values obtained are compared with thoseoriginating from untreated cells or undergoing a reference treatment(IL-1, estradiol, calcic inhibitor, for example).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the results of Example 1.

FIG. 2 is a graph of the results of Example 2.

FIGS. 3 and 4 are graphs of the results of Examples 3 and 5,respectively.

EXAMPLE I

Fixing the Calcium by the Line of Osteoblasts G 292 Compared withEstradiol, Vitamin D3 and Extract of Padina (EPP) in the Presence or notof Interleuking 1.

E2/ EPP + D3 + Controls IL-1 E2 IL-1 EPP IL-1 D3 IL-1 Osteo- 1,000 0,2001,843 0,200 2,659 2,785 1,215 0,125 blasts G292 1,000 0,070 2,648 0,0700,000 2,485 1,540 0,070 The FIG. 1 schematizes the results obtainedunder these conditions.

The cells treated solely by interleukin IL-1 have a level of inactivitymuch lower than that of the control cells. The cells treated by IL-1 andthe extract of Padina have an activity bordering on that of the controlcell.

EXAMPLE II

Calibration of the Extract of Padina with Estradiol in the Presence ofIL-1

Standard Average Deviation 50% Control 1.194 0.182 ED = 0.755 IL-1 0.3150.002 ED = Effective dose 100 ng EPP + IL-1 0.663 0.016 200 ng EPP +IL-1 0.671 0.129 500 ng EPP + IL-1 1.139 0.196 1000 ng EPP + IL-1  1.1530.216 Estr + IL-1 0.357 0.013 Est 10⁻⁹ 0.706 0.102 Est 10⁻⁸ Est 10⁻⁸1.115 0.167 Est 10⁻⁷ 1.151 0.137 FIG. 2 schematizes the resultsobtained.

240 ng EPP=10⁻⁹ M Estradiol=272 μg/L i.e. 272 ng/ml. The dose of 240 ngof extract of Padina corresponds with the activity of 10⁻⁹ M estradiolon the fixing of calcium (240 ng of extract of Padina=272 μg/l ofestradiol, i.e. 272 ng/ml). We may also say that 240 ng of extract havea titre of one unit.

EXAMPLE III

Calibration of the Extract of Padina (EPP) and Estradiol on the Fixingof Calcium in Relation to the Effect of a Calcic Inhibitor such asVerapamil.

Standard Average Deviation 50% Control 1.194 0.182 ED = 0.789 Verap 10μg 0.383 0.018 ED = Effective dose 100 ng EPP + Verap. 0.907 0.081 200ng EPP + Verap. 1.189 0.345 500 ng EPP + Verap. 1.222 0.173 1000 ngEPP + Verap.  1.249 0.140 Est + Verap. 0.373 0.023 Est 10⁻⁹ 0.706 0.102Est 10⁻⁸ 1.115 0.167 Est 10⁻⁷ 1.151 0.137 The FIG. 3 schematizes theresults obtained. Verap. = Verapamil and Est. = Estradiol

EXAMPLE IV

Variation of the Extract of Padina in Relation to Estradiol and a CalcicInhibitor: Inchonine

Standard Average Deviation 50% Control 1.194 0.182 ED = 0.627 Cinchonine10 μg 0.059 0.003 EPP = Extract 100 ng EPP + Cinchonine 0.981 0.272 ofPadina 200 ng EPP + Cinchonine 1.163 0.147 Est = Estradiol 500 ng EPP +Cinchonine 1.100 0.246 ED = Effective 1000 ng EPP + Cinchonine  1.1230.194 dose     Estr + Cinchonine 0.357 0.013 Est 10⁻⁹ 0.706 0.051 Est10⁻⁸ 1.115 0.084 Est 10⁻⁷ 1.151 0.068

EXAMPLE V

Variation of Bone Density of the Lumbar Column in the Course of a Year

Average time Average variation of patients Time N = 8 treated with 200mg of EPP, N = 8 0 0.000 0.000  3 months −0.230 0.515  6 months −0.5201.355  9 months −0.725 1.510 12 months −0.845 1.725 FIG. 4 schematizesthe results obtained.

The data on the activity are compared using statistical tests comparingof two averages and two standard deviations (student test). The resultsare all statistically significant.

Examples 1 to 5 show the agreement of the results obtained by theprocess according to the invention with the results obtained bybiological means and human clinical trials. The few differencesdiscovered (240 ng per 272 ng/ml of estradiol) are principally due tothe uncertainty of measures necessary for the evaluation of a biologicalprocess.

It may therefore be considered that the biological extracts of Padinapavonica have an activity comparable with that of estradiol, but that inno case is it affected adversely by the presence of interleukin or aninhibitor of the calcic canals such as Verapamil or Diltiazem, unlikethe calcium fixing agents already known such as estradiol, vitamin D andcalcitonine.

1. A method of determining the calciotropic activity of a preparation ofalgae of the genus Padina pavonica, said preparation selected from thegroup consisting of an extract of said algae and a powder of said algae,said method consisting of culturing human or animal osteoblasts in aculture medium rich in calcium ions in a well plate, adding to saidculture medium said extract or a powder of said algae Padina pavonicaand culturing between 24 to 72 hours, thereafter eliminating non-fixedcalcium ions in the culture medium, acidifying the culture medium todestroy extracellular matrix formed by the osteoblasts, thereafterfiltering the culture medium to remove solid material, and determiningconcentration of fixed calcium ions in the osteoblasts in comparisonwith a calibration scale of a known activator of the fixation of calciumor in the presence of an inhibitor of the fixation of calcium or both.2. The method of claim 1, wherein results of calculation of calciotropicactivity are expressed in comparison with results obtained with adetermined amount of an active compound known to promote calcium uptakeby the osteoblasts.
 3. The method of claim 1, wherein results ofcalculation of calciotropic activity are expressed comparatively to theknown effects of a determined amount of estradiol.
 4. The method ofclaim 1, wherein the osteoblasts are in suspension having a cell densityas determined by turbidimetry or by cell density calculation based on areference value.
 5. The method of claim 1, wherein the culturing furthercomprises adding culture medium and a deleterious agent inhibiting thefixing of calcium to each well of said well plate, thereafter a setvolume of dispersion of said extract or powder of algae to be tested isadded to the medium in said well plate, and the medium is incubated at37° C. in an atmosphere of carbon dioxide for one to three days.
 6. Themethod of claim 1, wherein estradiol is used as the known activator ofthe fixation of calcium.
 7. The method of claim 1, wherein the inhibitorof the fixing of the calcium is a pro-inflammatory agent.
 8. The methodof claim 7, wherein the pro-inflammatory agent is interleukin IL-1. 9.The method of claim 1, wherein the inhibitor of the fixing of thecalcium is an agent which blocks the calcium channels.
 10. The method ofclaim 1, in which the inhibitor of the fixing of the calcium is an agentwhich blocks the calcium channels selected from the group consisting ofVerapamil and Cinchonine.